Electrospun nanofibers have been utilized in the field of tissue engineering for over a decade and the technology of electrospinning and the nanofibers themselves are advantageous for several reasons: nanofiber production through electrospinning is relatively simple, the mats generated by the nanofibers can closely resemble the three-dimensional structure of the extracellular matrix of certain tissues, and parameters of the electrospinning technique can be altered to yield a final product of specific structure and function. The latter is a unique quality of electrospinning and a principal factor in utilizing the method for tissue engineering applications.
Typically, a synthetic, biodegradable polymer, such as poly L-lactic acid (PLLA), polyglycolic acid (PGA), or polycaprolactone (PCL), is dissolved in an appropriate solvent to generate a moderately viscous solution which is suitable for electrospinning. The design of the collector for the electrospinning device can have a marked effect on the morphological characteristics of the nanofibrous mat produced. For instance, it is known that rotating collectors may be employed to obtain aligned nanofibers suitable for engineering nerve tissue. In most instances, however, the nanofibrous mat is collected onto a smooth surface, resulting in a thin, two-dimensional mat or “sheet” of nanofibers.
These two-dimensional mats of nanofibers have demonstrated notable benefits in repairing damage to tissues such as skin, which lack the need for a more complex, macroscopic three-dimensional structure. Much of the more recent research in tissue engineering with nanofibers has been focused primarily on the modification and manipulation of the fibers at the microscopic level. While these approaches identify various areas for potential improvement in the field of tissue engineering, they remain of limited utility in applications where macroscopic three-dimensional structures are required to regenerate larger tissues and organs.
Production of nanofibers for three-dimensional scaffolds has been more problematic. One approach for producing three-dimensional tubes of nanofibers which could be appropriate for tissue engineering blood vessels involves introducing a third dimension to the fibrous mat by rolling two dimensional nanofiber sheets into simple, hollow tubes. This approach has not been an effective method for producing tissue scaffolds of greater three-dimensional complexity. Others have fabricated heart valve prostheses from PCL using electrospinning. And while the trileaflet shape of the valve demonstrated a significant increase in overall three-dimensional complexity, the prosthesis remained relatively thin and this approach is unsuited to applications requiring increased thickness and strength of the scaffold.
Another approach known in the art is to produce nanofibers having overall three-dimensional structure resembling a cotton ball. Such scaffolds are known as Focused, Low density, and Uncompressed nanoFibrous (FLUF) mesh. These “cotton ball”-like FLUF scaffolds have a lower density of fibers when compared to flat nanofiber mats and have been found to provide a suitable three-dimensional environment for the infiltration and growth of INS-1 cells. However, these “cotton ball” scaffolds do not exhibit the necessary strength or structure for the engineering of other cells and tissues, such as those from bone and additional connective mineralized tissues. Yet another approach involves fabricating electrospun scaffolds using solutions of alginate and poly-ethylene oxide (PEO), which produce three-dimensional structures because of charge repulsion between individual fibers as a result of the negatively charged alginate. As with the FLUF scaffolds, however, the alginate-PEO scaffolds lack sufficient mechanical strength to be effective for engineering of harder tissues such as bone and other calcified tissues.
Recently, researchers have successfully designed and developed tissue-engineered bone in the shape of human digits (phalanges). Their engineered models consisted in part of the thin tissue (periosteum) covering the long bones from young calves. Periosteum was dissected and then wrapped and sutured about biodegradable polymer scaffolds composed of PCL/PLLA (75/25) and shaped like human digits. The periosteum/scaffold constructs were then implanted in athymic, immunodeficient mice (lacking the means for rejecting foreign tissue such as that from calves and other non-mouse species) for 20 and 40 weeks. Constructs retrieved from the mice at various time intervals of implantation and development demonstrated that bone tissue could be reproducibly regenerated in three dimensions by utilizing the periosteum as a viable source of bone progenitor cells. Further, it was found that the addition to constructs of certain growth factors, such as osteogenic protein-1 (OP-1) and basic fibroblast growth factor (bFGF), can expedite cell proliferation and differentiation. These molecules, applied directly to cells or provided to them through release and delivery vesicles or other means, have been shown to lead to more rapid formation of bone and other tissues.
In addition to such things as growth factor addition, the use of electrospun nanofibers has been shown to result in increased cell attachment and proliferation when compared to cells cultured in a monolayer environment. In this context, researchers have attempted to incorporate nanofibers into their experimental digit designs by wrapping the PCL/PLLA scaffolds with thin, pre-formed sheets of PGA nanofibers prior to application of periosteum. These experiments, however, have been largely unsuccessful because of the difficulty in maintaining direct contact at the interface between the nanofibers and the underlying PCL/PLLA scaffold. It has been found that without direct contact between the tissue scaffold and periosteum, the osteoprogenitor cells cannot infiltrate the scaffold and grow. Alternatively, suturing mats of nanofibers, rather than wrapping them, to the scaffolds as an alternate approach to direct contact is time-consuming and requires expertise to produce a suitable nanofiber-covered scaffold and subsequent construct. Additionally, as the complexity of the underlying construct increases, so does the number of sutures needed to ensure the nanofiber sheet remains in close contact with it.
A simple and novel method to circumvent the difficulties in designing an intimate contact between a nanofiber and scaffold is to apply nanofibers directly to the surface of the polymer scaffolds utilizing the electrospinning process. The surface to be coated needs only to be grounded, and thereby made electrically conductive, and placed in the path of the electrospun nanofibers as they are produced. Naturally conductive materials are easily coated, but poorly conductive materials, such as PCL/PLLA or other such polymers typically used for making polymer scaffolds have been found to be much more difficult to coat. Initial attempts to coat these polymer scaffolds by placing PCL/PLLA (75/25) scaffolds onto a flat, grounded electrical collector, directly in the path of the nanofiber jet were largely unsuccessful, resulting in only a few nanofibers being deposited onto the surface of the scaffolds.
What is needed in the art is a method and apparatus to facilitate and improve efficient application of nanofibers to the surface of other PCL/PLLA polymer scaffolds by providing a much more direct connection between the scaffold and the grounded plate collector while allowing the scaffold to be supported above the collector in a manner which promotes nanofiber deposition over the top and sides of the PCL/PLLA scaffold, closely covering the scaffold surfaces.